This program is designed to fill the need for a versatile model to investigate development of the human brain as affected by HIV-1 exposure and infection. We propose to develop an alternative to primary human neural stem cells as the system for study of neural cell development and neuropathogenesis during HIV infection. Our immediate goal is to induce neuronal differentiation in a well characterized human cell line using physiological cues present in the developing brain through coculture with fetal brain and then to evaluate the influence of HIV-1 upon these cell-cell interactions. Our long-term goal is to apply gene targeting technology to test the function of specific genes in HIV-1 neuropathogenesis using this model system. The Specific Aims are: 1) To test the abillty of NT2 cells to differentiate in response to cues provided by primary brain cells during aggregation and maturation. 2) To test the effects of HIV-1 exposure on NT2 development within aggregates. 3) To determine whether gene targeting methods can be applied to affect NT2 interaction with HIV-1 and with other neural cells. Fetal brain aggregates and monolayer cultures will be established with NT2 and the development of both primary cells and NT2 will be monitored by immunocytochemistry. NT2 will be infected with X4 or R5 HIV prior to or during culture with primary cells and cytopathic and cytotoxic effects will be monitored. The coding region of CXCR4 in NT2 will be ablated by insertion of genes encoding selectable markers and this "knock out" NT2 will be tested in the coculture system for modulation of the response to HIV or products of HIV infected cells. These studies will begin construction of a new system for analysis of human gene involved in HIV neuropathogenesis. Ultimately, results obtained in this system can be confirmed in primary cells or by evaluation of the expression of specific gene products in the HIV-infected brain.